L60: a novel monoclonal antibody

ABSTRACT

L60 is a novel monoclonal antibody that will react with Leu 22. It is equally reactive in formalin, ZnSO 4  or Bouin&#39;s fixed, paraffin-embedded tissues as well as in frozen tissues.

This is a continuation of application Ser. No. 139,491, filed Dec. 30,1987,

which issued as U.S. Pat. No. 4,945,056 on July 31, 1990.

FIELD OF THE INVENTION

The present invention relates to the detection of the Leu 22 antigen,and more particularly, relates to the use of a novel monoclonal antibody(L60) which will bind to the Leu 22 antigen in frozen and in formalin,ZnSO₄ and Bouin's fixed, paraffin-embedded tissues.

BACKGROUND OF THE INVENTION

The vast majority of leukocyte antigens are destroyed by routineprocessing to paraffin blocks. As a result, the use of monoclonalantibodies (MAbs) to identify cell lineage, cell differentiation andcell subset populations have been restricted to fresh frozen tissuesections. In many instances, however, fresh frozen tissue sections maynot be available for analysis. It is desirable, therefore, to find andidentify MAbs that will react with leukocyte antigens in routinelyprocessed, paraffin-embedded tissues.

Currently on the market are several monoclonal antibodies purportedlyconstructed to satisfy this desire. Among these antibodies are: ClonabMT1, MT2, MB1 and MB2 (available from BioTest, Inc.); LN1, LN2 and LN3(available from Techniclone International); and Dako LC and UCHL-1(available from Dako Corp.). Descriptions of each group of monoclonalantibodies have been reported in: Poppema et al., Monoclonal Antibodies(MT1, MT2, MB1, MB2,) Reactive with Leukocyte Subsets inParaffin-Embedded Tissue Sections, Am. J. Pathol., 127:418-429 (1987);Epstein et al., Two New Monoclonal Antibodies (LN-1, LN-2) Reactive inB5 Formalin-Fixed, Paraffin-Embedded Tissues with Follicular Center andMantle Zone Human B Lymphocytes and Derived Tumors, J. Immunol.,133:1028-1036 (1984); and Norton et al., Momoclonal Antibody (UCHL1)that Recognizes Normal and Neoplastic T Cells in Routinely FixedTissues, J. Clin. Pathol., 39:399-405 (1986).

Although these MAbs function in other than frozen tissue sections, theantigens they react with are of limited use both in terms of theirdistribution and specificity. A number of these MAbs will cross-reactbetween T and B cell lines, for example. Thus, the usefulness of theseMAbs is somewhat limited.

It would be desirable, therefore, to find an antigen that is distributedon T cell malignancies but not on B cell malignancies. This is ofclinical importance because T cell malignancies behave more aggressivelythan B cell malignancies, and thus, require more aggressive treatment.

Similarly, the diagnosis of Hodgkin's from non-Hodgkin's lymphomas isimportant because of the vast prognostic and therapeutic differencesbetween the diagnostic alternatives. For determination of Hodgkin'sversus non-Hodgkin's lymphomas, therefore, it would be useful to have amonoclonal antibody that would identify an antigen that discriminatesbetween Hodgkin's and non-Hodgkin's lymphomas.

Leu 22 is antigen of approximately 100 kD that is found on normal andmalignant T cells, peripheral blood monocytes and a subpopulation ofnormal B cells and tissue macrophages. It is conserved on non-Hodgkin'slymphomas of T cell origin but is not found on Reed-Sternberg cells inHodgkin's disease. Thus, the Leu 22 antigen has many of thecharacteristics desired.

BRIEF DESCRIPTION OF THE INVENTION

L60 is monoclonal antibody that recognizes the Leu 22 antigen on Tcells, blood monocytes, tissue macrophages and on a subpopulation of Bcells. It was derived from a mouse hybridoma clone DB38.3E3. Thehybridoma was formed between spleen cells from Balb/c mice immunizedwith phytohemagglutinin(PHA) activated T cells and the murineplasmacytoma cell line SP2/0 AG 14. L60 is an IgG_(1k) type monoclonalantibody. L60 reacts with T lymphomas fixed in either formalin, ZnSO₄ orBouin's fixative. It is useful in distinguishing Hodgkin's fromnon-Hodgkin's lymphomas.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a rendering of an autoradiograph of a 10% SDS/PAGE gel whereinHPB-ALL cells were reacted with Anti-Leu 22 or with one of severalcontrols or L60 like clones by the lactoperoxidase/glucose oxidasemethod and were labeled with ¹²⁵ I. Material then was applied to the geland run as follows: Lane 1--gamma 1 control; Lane 2--Anti-Leu 2a; Lane3--Anti-Leu 2; Lane 4--Anti-Leu 2c; Lane 5--Anti-Leu 22 (supernatantfrom mouse culture); Lanes 6-8 and 10-11--L60 like clones (L60.1, 7D12,7D12.12, L50 and L51 respectively); Lane 9--1G10 (a gamma 2a, L60 likeclone); and Lane 10--1G11. Lanes 13 and 14 were not used.

DETAILED DESCRIPTION OF THE INVENTION

The L60 monoclonal antibody was prepared as follows. T lymphocytes wereisolated from human peripheral blood by density dependent centrifugationon Ficoll-Hypaque (Litton Bionetics). T cells were cultured in RPMI 1640(GIBCO) and 10% fetal calf serum (FCS) to which 1.0% PHA was added.After 72 hours, 1×10⁷ PHA-activated T cells were injectedintraperitoneal ("I/P") into a Balb/c mouse. On day 19, the mouse wasboosted I/P with 1×10⁷ PHA-activated T cells. On day 78, the mouse wasboosted with 4×10⁶ PHA-activated T cells intravenous ("I/V").

On day 81, the mouse was sacrificed and the spleen excised. Splenocytesthen were fused with the continuous plasmacytoma fusion partner SP2/0 Ag14, Shulman et al., Nature, 276:269 (1978), in 35% polyethylene glycol.Similar procedures for preparations of spleen cells and fusion have beenpreviously described in U.S. Pat. Nos. 4,172,124 and 4,196,265 which areincorporated herein by reference.

Viable cells resulting from the fusion process were plated in 96 wellmicroculture plates (B-D Falcon) in Dulbecco's Modified Eagle's Medium(GIBCO) containing 20% fetal calf serum ("FCS"). A solution of 15 mMHEPES (GIBCO) and azaserine-hypoxanthine (2 ug/ml and 10⁻⁴ M finalconcentration respectively) was added to each well as a selection mediaaccording to the procedures of Buck et al., Production of HumanMonoclonal Antibodies, in Monoclonal Antibodies and Functional CellLines, Plenum Publishing Corp. (Kennett et al., eds) (1984). hybridomasgrown in this medium may be separated from unfused myeloma cells andspleen cells which die shortly after fusion. Incubation continued for7-10 days or until visible colonies appeared.

Hybridoma supernatants initially were characterized by binding toPHA-activated T cells and non-binding to the LB B cell line (alymphoblastoid cell line) using a Pandex™ screening device (PandexLaboratories, Inc.). 1×10⁵ PHA-activated T cells or LB B cells wereadded to each well of a 96 well Pandex™ plate. To each well was added 30ul of supernatant. After 30 minutes, the cells were washed in 0.15Mphosphate buffered saline (PBS). Goat anti-mouse Ig antibody coupled tofluorescein isothiocyanate (GAMIg-FITC, Becton Dickinson ImmunocytometrySystems) then was added to each well, incubated and the results wereread on the Pandex screening device.

Cells from those wells whose supernatants tested positive were replatedin 24 well microculture plates and grown in Dulbecco's Modified Eagle'sMedium (M.A. Bioproducts) containing hypoxanthine and 20% FCS.Supernatants from each well were added to wells containing eitherPHA-activated T cells or LB B cells. GAMIg-FITC again was added. Stainedcells from these wells were subjected to analyses by flow cytometryinstrumentation (FACScan™, Becton Dickinson Immunocytometry Systems).Cells were examined for forward and right angle light scatter andfluorescence. From the analyses, clone DB38.3E3 was selected. It hasbeen deposited with American Type Culture Collection, Rockville, Md.,under the terms of the Budapest Treaty on Oct. 20, 1987 as accessionnumber HB-9575.

The monoclonal antibody produced by this clone has been designated L60.It also may be referred to as Anti-Leu 22. It has an isotype ofIgG_(1k). It will immunoprecipitate an antigen on mitogen activated orresting T cells of approximately 100 kD under reducing conditions. SeeFIG. 1. This antigen has been designated Leu 22. It is not, however, theT-200 antigen. Immunoperoxidase staining of normal tissue shows that L60strongly reacts with T cells and macrophages in tonsil, cortical andmedullary thymocytes in thymus, and T cells in spleen.

To demonstrate its ability to identify Leu 22 in fixed,paraffin-embedded tissues, L60 was reacted with a variety of tissuespecimens and detected using a two-step indirect staining technique.Tissue sections were fixed in formalin, ZnSO₄ or Bouin's and embedded inparaffin in accordance with standard procedures. The tissues had beenexcised from patients with a variety of lymphomas.

5 um paraffin tissue sections were cut and floated on a water bathcontaining gelatin and chromium potassium sulfate. The sections thenwere applied to slides and dried. The sections then were deparaffinizedby placing them in 4 changes of Histoclear™ (National Diagnostics),followed by several changes in a graded ethanol series, distilled waterand 0.1M phosphate buffered saline (PBS). Routinely, sections were nexttreated to block endogenous peroxidase by placing the section in 3%aqueous H₂ O₂ for 5 minutes. Sections then were rinsed in 0.1M PBS.After rinsing, sections were incubated with the approriate monoclonalantibody in a humified chamber to avoid drying. After incubation, thesections were rinsed in PBS.

At this stage, it is important to add a blocking serum. A 1:10 dilutionof normal goat serum was applied for 40 minutes. Excess serum wasremoved by wiping around the section only. Rinsing is to be avoided.

Once the excess serum is removed, horseradish peroxidase conjugated goatanti-mouse IgG (Vector) in PBS containing 0.1% thimerosal (Sigma), 0.2%gelatin and 5% normal human serum is applied for 40 minutes. The sectionagain was rinsed in PBS as above.

Alternatively, biotin conjugated horse anti-mouse IgG followed byhorseradish peroxidase conjugated avidin (Vector) may be used in a threestep procedure in place of the two step secondary antibody enzymecombination.

Once the sections have been incubated with the horseradish perixidaseconjugate, enough 3.3 Diaminobenzidine (DAB, Sigma) in 10 ul of 30% H₂O₂ and 1 ml of 0.1 PBS was added to cover the section for 5 minutes. Thesection then was rinsed in PBS as above.

Once rinsed, the section was treated with a 0.5% solution of CuSO₄ in0.85% NaCl for 5 minutes. The section then was rinsed in distilledwater. The section then was counterstained in Gill's ProgressiveHematoxylin (4.0 gm hematoxylin [Sigma], 0.4 gm NaIO₃, 70.4 gm Al₂(SO₄)₃ 18H₂ O, 20 ml glacial acetic acid) for 30 seconds, and rinsed intap water.

The section then was dipped in a saturated solution of aqueous lithiumcarbonate until blue. The section again was rinsed in tap water, anddehydrated in alcohol, Histoclear™ and xylene and coverslipped in aresinous mounting media. The section then was ready for microscopeexamination.

If frozen sections were used, 4-5 um cryostat sections were air driedfor 2 hours. The sections must be thoroughly dry. Dried sections wereapplied to slides pretreated with a chrom alum-gelatin solution, andwere then fixed in reagent grade acetone at room temperature for 10minutes then air dried. The sections then were stained immediately asabove, with the exceptions that counterstaining should be done for 1minute and dipping in lithium carbonate was omitted. If the sectionswere not used immediately, they were stored at -70° C. with a desiccant.Stored sections should come to room temperature in the presence of adesiccant prior to staining.

Table 1 sets forth the lymphomas examined using the above procedures.The lymphoma type was determined in accordance with standard pathologicprocedures. The number of lymphomas examined and reactive with L60 isgiven. "ND" indicates that the results were not determined. As evidentfrom Table 1, L60 is reactive in several paraffin-embedded tissues fromT cell but not B cell lymphomas.

                  TABLE 1                                                         ______________________________________                                        L60 Positive                                                                                Lymphoma Type                                                   Fixative        T Cell  B Cell                                                ______________________________________                                        Bouin's         6/8     ND                                                    Formalin        5/5     0/5                                                   ZnSO.sub.4      5/11    ND                                                    ______________________________________                                    

Variations and modifications of the above described invention maysuggest themselves to those skilled in the art. Accordingly, thedescription should not be taken in any limiting sense.

What is claimed is:
 1. A monoclonal antibody capable of reacting withthe same antigen as that recognized by an antibody produced by thehybridoma deposited as ATCC HB-9575.